Numerous epidemiological studies in various populations have shown that consumption of substantial amounts of fruits and vegetables rich in carotenoids can reduce the risk of acquiring several types of cancers. As a result, scientists have been focusing on investigating the protective effect of carotenoids such as beta-(β-)carotene in prevention of cancer, cardiovascular and eye diseases. These studies have been carried out despite the fact that β-carotene is only one of the prominent carotenoids found in fruits and vegetables whose consumption has been associated with health benefits. The reasons for such focus can be attributed to the pro-vitamin A activity of β-carotene and the limited commercial availability of other prominent food carotenoids.
Among the 40 to 50 carotenoids that are available from the diet and may be absorbed, metabolized, or utilized by the human body, only 13 carotenoids and 12 of their stereoisomers are routinely found in human serum and milk. [See Khachik et al., Anal. Chem., 69:1873-1881 (1997).] In addition, there are 8 carotenoid metabolites and one stereoisomer in human serum or plasma that result from a series of oxidation-reduction reactions of three dietary carotenoids: lutein, zeaxanthin and lycopene. These metabolites were first isolated and characterized by Khachik et al. [See Khachik et al., Anal. Chem., 64:2111-2122 (1992).]
In another study, the ingestion of purified supplements of dietary (3R,3′R,6′R)-lutein and (3R,3′R)-zeaxanthin was shown to not only result in an increase in the blood levels of these compounds in humans, but also in an increase in the concentration of their oxidative metabolites in plasma. [See Khachik et al., J. Cellular Biochem., 22: 236-246 (1995).] These findings provided preliminary evidence that carotenoids may function as antioxidants in disease prevention. In addition, these results also established the importance of non-vitamin A-active dietary carotenoids, particularly, lutein, zeaxanthin, and lycopene.
There is increasing evidence that the macular pigment carotenoids, lutein and zeaxanthin, may play an important role in the prevention of age-related macular degeneration (ARMD), cataract formation, and other light-induced oxidative eye damage. In 1985 and 1993, Bone et al. demonstrated that the human macular pigment is a combination of lutein and zeaxanthin, and speculated that these dietary carotenoids may play a role in the prevention of an eye disease ARMD. [See Bone et al., Vision Research, 25:1531-1535 (1985) and Bone et al., Invest. Ophthalmol. Vis. Sci., 34: 2033-2040 (1993).] Further work in a case-controlled epidemiological study in which the high consumption of fruits and vegetables, rich specifically in lutein and zeaxanthin was correlated to a 43 percent lower risk of ARMD later confirmed that speculation. [See Seddon et al., J. A. Med. Assoc., 272(18) 1413-1420 (1994).] It has also been reported that an increased level of serum carotenoids other than β-carotene is associated with a lower incidence of heart disease. [See Morris et al., J. Amer. Med. Assoc., 272(18):1439-1441(1994).]
Bernstein et al. identified and quantified the dietary carotenoids and their oxidative metabolites in all tissues of the human eye and reported that nearly all ocular structures examined with the exception of vitreous, cornea and sclera had quantifiable levels of dietary (3R,3′R,6′R)-lutein, zeaxanthin, their geometrical (E/Z) isomers, as well as their metabolites, (3R, 3′S,6′R)-lutein (3′-epilutein) and 3-hydroxy-beta,epsilon-caroten-3′-one. In the iris, these pigments were thought likely to play a role in filtering phototoxic short-wavelength visible light and to act as antioxidant in the ciliary body. Both mechanisms may be operative in the retinal pigment epithelium/choroid (RPE/choroids). [See Bernstein et al., Exp. Eye Research, 72(3):215-223 (2001].]
A study of the distribution of macular pigment stereoisomers in the human retina identified (3S,3′S)-zeaxanthin in the adult retina, particularly in the macula. It was proposed that dietary lutein and zeaxanthin are transported into an individual's retina in the same proportions found in the blood serum, although the two pigments are present in the eye in ratios different from those found in the blood. Thus, zeaxanthin predominates over lutein by a ratio greater than 2:1 in the foveal region, with the macular pigment optical density dropping by a factor of 100 and the zeaxanthin to lutein ratio reversing to about 1:2. [See Bone et al., Invest. Ophthalmol. Vis. Sci., 29:843-849(1988).] Some lutein is converted into the non-dietary meso-zeaxanthin primarily in the macula. [See Bone et al., Exp. Eye Res., 64(2): 211-218 (1997).] Such reports lend support to the critical role of ocular carotenoids, and therefore to the importance of commercial production of dietary carotenoids in general, and particularly lutein and zeaxanthin.
The Tagetes genus is a member of the family Compositae, alternatively known as Asteraceae, and comprises some thirty species of strongly scented annual or perennial herbs. Tagetes are native from Arizona and New Mexico to Argentina. [See Hortus Third A Concise Dictionary of Plants Cultivated in the United States and Canada, MacMillan Publishing Company (1976).] Cultivated genera include Tagetes erecta, commonly referred to as African marigold, Tagetes patula, commonly referred to as French marigold, Tagetes erecta×patula, commonly referred to as Triploid marigolds, and Tagetes tenuifolia also known as Tagetes signata or signet marigold.
A marigold inflorescence is a solitary head comprised of a dense cluster of several hundred sessile or subsessile small flowers also known as florets. Marigolds have radiate flower heads with outer ray florets that are ligulate or strap-shaped around the central tubular shaped disk florets. Some forms of marigold flower heads have most of their disk flowers transformed into ray flowers and contain few, if any, disk flowers. Such flower heads are referred to as double-flowered.
The ray flowers or florets are often referred to as petals by lay persons who may also refer to the flower heads as flowers. For ease of understanding, marigold flower heads will be referred to herein as flowers or flower heads, whereas the flower head-component flowers or florets, stamens, stigmas and carpels will be referred to as petals.
Cultivated marigolds possess showy flowers and are useful for ornamental purposes. In addition, the genus is recognized as a source for natural colorants, essential oils, and thiophenes. Dried marigold petals and marigold petal concentrates obtained from so-called xanthophyll marigolds are used as feed additives in the poultry industry to intensify the yellow color of egg yolks and broiler skin. [See Piccalia et al., Ind. Crops and Prod., 8:45-51 (1998).] The carotenoids desired in poultry tissues are a function of their dietary concentration, because poultry do not have the ability to synthesize carotenoids de novo. [See Balnave et al., Asian-Australiasian J. Animal Sci., 9(5):515-517 (1996).]
Xanthophyll marigolds differ in several characteristics from ornamental marigolds. First and foremost, xanthophyll marigolds are used as an extractable source for carotenoids and have plant habits that differ from ornamental marigolds. Ornamental marigolds typically grow only about 45 to about 60 cm from the ground, whereas xanthophyll marigolds grow to about 65 to about 70 cm from the ground. Xanthophyll marigolds grow in a bushier habit than do ornamental marigolds, and can be grown as row crops whereas ornamental marigolds typically cannot. Xanthophyll marigolds are typically dark orange in color, whereas ornamentals can be white, yellow, or orange in color, or can have mixed colors, including mahogany colors due to the presence of anthocyanin pigments.
The pigmenting ability of marigold petal meal resides largely in the oxygenated carotenoid fraction known as the xanthophylls, primarily lutein esters. [See Piccalia et al., Ind. Crops and Prod., 8:45-51 (1998).] The xanthophyll zeaxanthin, also found in marigold petals, has been shown to be effective as a broiler pigmenter, producing a highly acceptable yellow to yellow-orange color. [See Marusich et al., Poultry Sci., 55:1486-1494 (1976).] Of the xanthophylls, the pigments lutein and zeaxanthin are the most abundant in commercially available hybrids. Structural formulas for lutein and zeaxanthin are shown below. 
Each of lutein and zeaxanthin contains one hydroxyl group in each of their terminal ring structures, so that each molecule contains two hydroxyl groups. Lutein is believed to be biologically produced by two separate hydroxylations of α-carotene, whereas zeaxanthin is believed to be biologically produced by two separate hydroxylations of β-carotene.
Both α-carotene and β-carotene are understood to be formed by the action of appropriate cyclase enzymes on lycopene to first yield δ-carotene or γ-carotene that thereafter cyclize further to form α-carotene or β-carotene, respectively. Lycopene, γ-carotene, α-carotene and β-carotene are each hydrocarbon carotenoids that are referred to in the art as carotenes. Thus, carotenoid pigments can be grouped into one or the other of two families: the hydrocarbon carotenes or the oxygenated xanthophylls. Phytoene, the first C40 carotenoid in the pathway, is a colorless hydrocarbon. The hydrocarbon carotene pigments with the exception of β-carotene typically do not accumulate in marigold leaves or flower parts, whereas the xanthophylls do accumulate in both leaves and flower parts.
FIG. 1 shows a schematic representation of the biological synthesis pathway for the production of lutein and zeaxanthin and later products from phytoene via lycopene, γ-carotene, α-carotene and β-carotene. Lutein and zeaxanthin are present in marigold petals primarily as mono- and di-esters of fatty acids. FIG. 1 also notes epoxide-containing later products that can arise from zeaxanthin, of which violaxanthin is an intermediate in the abscisic acid biosynthetic pathway.
For the feed additive industry, xanthophyll marigolds are produced primarily in Mexico, Peru, Africa, India, China and Thailand. Modern, commercial varieties include ‘Orangeade’, one of the original xanthophyll producing varieties, and commercial improvements of ‘Orangeade’, including ‘Deep Orangeade’ having larger flowers and greater pigment yields, and ‘Scarletade’ an improvement for xanthophyll concentration. Thus, ‘orangeade’ is reported to contain xanthophylls at about 9-12 mg/g of dry whole flower heads (including calyx), ‘Deep Orangeade’ is reported to have about 10-13 mg/g of those pigments, and ‘Scarletade’ is said to contain about 12-18 mg/g of xanthophyll pigments in dry flower heads weighed with the calyx. These varieties are available from PanAmerican Seed Co., 622 Town Road, West Chicago, Ill. 60185.
Whereas lutein is the major xanthophyll in marigold flowers, some current varieties yield extract products with zeaxanthin ratios [zeaxanthin/(lutein+zeaxanthin)] typically in the 3 to 5 percent range (See Product Profile, Kemin Foods L.C., 600 E. Court Ave. Suite A, Des Moines, Iowa 50309). As is seen from the results hereinafter, zeaxanthin to lutein ratios obtained using ‘Scarletade’ are typically about 4 to about 7 percent.
Moehs et al., Plant Mol. Biol., 45:281-293 (2001) analyzed the biosynthesis of carotenoids in ornamental varieties of T. erecta, including a so-called wild type that had dark orange flowers, and plants with yellow, pale yellow and white flowers. Among other findings, those workers reported that although the different plants had a range in flower color from white (mutant) to dark orange, the differences in those flower colors were said to be due to the accumulation of very different amounts of the same carotenoid, lutein, rather than to accumulation of different carotenoid products or intermediates. The differences among the plants studied appeared to relate primarily to regulation of flux through the carotenoid pathway, rather than to the specific type of carotenoid produced or the accumulation of biosynthetic intermediates.
In addition, the so-called wild-type and mutant (white-flowered plant) leaves were reported to contain about the same relative quantity of carotenoid pigments, regardless of flower color. Those pigments were different from the pigments present in the petals. Thus, the only pigment reported for petals was lutein, whereas the leaves were reported to contain lutein as well as β-carotene, violaxanthin and neoxanthin. As is seen from FIG. 1, β-carotene but not lutein can be a precursor to the latter two pigments.
The Moehs et al., authors also compared the T. erecta genes they isolated with similar carotenoid-producing genes obtained from the leaves of Arabidopsis thaliana (Pogson et al., hereinafter). Identities between the gene products of about 70 to about 80 percent were reported at the protein level, with a higher level if putative plastid targeting signal peptides were excluded, and a lower level of identity at the DNA level. In leaves of A. thaliana, lutein is the predominant carotenoid, with β-carotene, violaxanthin and neoxanthin also being formed, but no zeaxanthin being normally accumulated.
Carotenoid biosynthesis in T. erecta is a complex system involving many genes and possibly two pathways. The impact of genetic mutations on carotenoid production cannot be predicted a priori. However, classic breeding techniques have produced “Orangeade”, ‘Deep Orangeade’ and ‘Scarletade’ T. erecta variants that produce the elevated levels of xanthophylls noted above. These relatively recently bred available varieties have not been subject to treatments that induce genetic mutations in an attempt to increase the zeaxanthin ratios.
Several workers have examined the effects of mutagens such as gamma irradiation, ethyl methanesulfonate (EMS) and nitrosomethylurea (NMU) on flowering plants, including marigolds. For example, Zaharia et al., Buletinul Institutului Agronomic Cluj-Napoca. Seria Agricultura 44(1): 107-114 (1991) reported on the chlorophyll-deficient effects of carotenoids in the coleoptile after seeds of Zinnia elegans, Tagetes erecta and Callistephus chinensis were irradiated with gamma irradiation in varying amounts. A paper by Geetha et al., Acta Botanica Indica, 20(2):312-314 (1992) reports on the chlorophyll deficient effects of gamma irradiation on Tagetes patula. 
Diaconu, Agronomie, 34(1):17-21 (1991) reported on the effects of EMS on germinating seeds from F2 polycrosses of commonly-called pot marigolds, or Calendula, that are not even of the genus Tagetes. Those workers noted a wide variation in flower color, inflorescence structure, yield and content of biologically-active substances in M2-M4 plants.
A study by Pogson et al., Plant Cell, 8:1627-1639 (1996) used EMS to mutagenize plants of Arabidopsis thaliana. This detailed study of 4000 M2 lines reported finding two loci in the carotenoid biosynthetic pathway in leaves that are involved with the production of lutein from γ-carotene. Those loci were referred to as lut1 and lut2. The lut2 locus was reported to be associated with the lycopene ε-ring cyclase enzyme, whereas the lut1 locus was reported to be associated with the lycopene ε-ring hydroxylase. Those workers noted (page 1631) that a decrease in lutein production was compensated for by an equimolar change in the abundance of other carotenoids, although only small amounts of those changes were due to an increased production of zeaxanthin.
Cetl et al., Folia Fac. Sci. Nat. Univ. Purkynianae Brun Biol., 21(1):5-56 (1980) reported extensive studies with T. erecta and other Tagetes species that from the meager descriptions appeared to all be ornamental varieties. Among those studies, those authors examined the effects of various concentrations of NMU on T. erecta seeds, and examined more than about 2000 plants. All M2 plants deviating from the phenotype of the parental cross were recorded, and M3 plants from M2 seeds of the phenotypically different plants were studied.
Those workers assayed plant height, plant diameter, flower head diameter and height of the flower head, as well as time to flowering, branching amount, branch length, cotyledon and leaf size, and flower stalk length. No mention is made regarding flower color or carotenoid levels in the leaves or petals.
Published PCT application WO 00/32788 of DellaPenna et al. asserts of a method of regulating carotenoid biosynthesis in marigolds. Those workers provide polynucleotide sequences said to be those that encode the lycopene β-ring cyclase and lycopene β-ring hydroxylase needed for the preparation of zeaxanthin from lycopene. Also disclosed is a lycopene ε-ring cyclase useful along with the lycopene β-ring cyclase for the preparation of α-carotene from lycopene. No teaching of the lycopene ε-ring hydroxylase needed for lutein production is provided.
Carotenoid biosynthesis is said in PCT application WO 00/32788 to be regulated by expression of a carotenoid synthesizing enzyme-encoding gene already present in marigolds such as those noted above, or by use of an anti-sense RNA encoded by such a nucleotide sequence provided. No evidence of such regulation is provided in the application. The phenomenon known as co-suppression by which the addition of a homologous gene causes both the native gene and transgene not to be expressed is not dealt with by those workers. [See for example, Fray et al., Plant Mol. Biol., 22:589-692 (1993) or Finnegan et al., Bio/Technology, 12:883-888 (September 1994).]
U.S. Pat. No. 6,383,474 to Soudant et al. teaches that phytoene and phytofluene, used in combination, are effective in preventing damage caused from oxidation and exposure to UV light. This combination is said to be useful as a topical preparation, as a pharmaceutical or as a food additive.
β-Carotene and lycopene are well-known food additives, with lycopene consumption recently being reported to provide a reduced risk of prostate cancer. [See, Giovannucci et al., J. Natl. Cancer Inst., 87(23):1767-1776 (1995).] Lycopene is naturally present as the red pigment in tomato skins, whereas β-carotene is the primary carotenoid pigment in carrots. Hauptmann et al. U.S. Pat. No. 5,618,988 teaches the preparation of carotenoid pigments such as β-carotene in storage organs of transformed plants such as carrots. Ausich et al. U.S. Pat. No. 5,858,700 teaches the isolation of lycopene crystals from an oleoresin as can be prepared from tomato skins. The structural formulas of lycopene and β-carotene are shown below. 
An increased ratio of zeaxanthin to lutein can provide an attractive substrate for biotechnological production of additional xanthophylls including the red xanthophyll, astaxanthin. Astaxanthin is widely used as a pigmenting agent for cultured fishes and shellfishes. The complete biomedical properties of astaxanthin remain to be elucidated, but initial results suggest that it could play an important role in cancer and tumor prevention, as well as eliciting a positive response from the immune system. [See Tanaka et al., Carcinogenesis 15(1):15-19 (1994); Jyonouchi et al., Nutrition and Cancer 19(3):269-280 (1993) and Jyonouchi et al., Nutrition and Cancer 16(2): 93-105 (1991).]
Astaxanthin supplied from biological sources, such as crustaceans, yeast and green algae is limited by low yield and costly extraction methods when compared with that obtained by organic synthetic methods. Usual synthetic methods however, produce by-products that can be considered unacceptable. It is therefore desirable to find a relatively inexpensive source of (3S,3′S) astaxanthin to be used as a feed supplement in aquaculture and as a valuable chemical for other industrial uses.
One approach to increase the productivity of astaxanthin production in a biological system is to use genetic engineering technology. Genes suitable for this conversion have been reported.
For example, Misawa et al. (See U.S. Pat. No. 6,150,130) specified DNA sequences including one isolated from the marine bacteria Agrobacterium aurantiacus sp. nov. MK1 or Alcaligenes sp. PC-1 that encodes a gene, referred to as crtW, used in the production of astaxanthin from zeaxanthin as a substrate by way of 4-ketozeaxanthin. Kajiwara et al. (See U.S. Pat. No. 5,910,433) identified a polynucleotide molecule, referred to as bkt, isolated from Haematococcus pluvialis that encodes a polypeptide having a beta-C-4-oxygenase activity for the production of (3S,3′S)astaxanthin from a host microorganism or a plant. In addition, Hirschberg et al. (See U.S. Pat. No. 5,965,795) described another DNA gene sequence from Haematococcus pluvialis, referred to as crtO, that encodes an enzyme that synthesizes astaxanthin from zeaxanthin by way of 4-ketozeaxanthin. Still further, Cunningham (See WO 99/61652) reported isolation of a DNA that encodes a protein having ketolase enzyme activity from Adonis aestivalis, a plant species having deep red flower color due in part to the accumulation of the ketocarotenoid astaxanthin.
It would therefore be useful if a marigold plant could be provided whose flower petals or leaves or both contain a commercially useful amount of xanthophylls and an altered ratio of lutein and zeaxanthin such that the usually reported 4 to about 7 percent zeaxanthin level were raised and the amount of lutein were decreased. It would also be useful if the ratios of other pigments could also be raised, and if such a plant had substantially the same phenotypical characteristics as a usual marigold plant grown adjacent to it. It would be further useful if a marigold could be produced that accumulated β-carotene or lycopene or both in the flower petals or leaves or both. The present invention provides several marigold plants, flower petals, leaves, seed that produces them, hybrids, oleoresins, mixtures of zeaxanthin and lutein, lycopene and β-carotene in proportions not normally found in marigolds, as well as comestible materials containing zeaxanthin, lutein, α-cryptoxanthin, antheraxanthin, neoxanthin and violaxanthin, lycopene, phytoene and β-carotene dissolved or dispersed in a comestible medium.